Proteins secreted via the general secretory (Sec) pathway play key roles in the pathogenesis of the many serious diseases caused by the Gram-positive pathogen Streptococcus pyogenes (group A streptococcus). Thus, an understanding of how polypeptides are exported via the Sec pathway and subsequently folded into their final three dimensional conformations will be critical to our understanding of how these proteins are delivered to their cellular targets, both on the host cell and on the streptococcal cell itself. Remarkably, very little is known about this essential process. A key question concerns the roles of targeting and accessory folding factors, and how the Sec pathway may be organized to coordinate interactions between folding factors and unfolded secreted polypeptide. The long-term goal of this project has been to address these issues using the SpeB cysteine protease as a model secreted virulence protein. Our identification of the requirement for several factors including RopA, HtrA, LuxS, GidA, Rpll and the novel factor RopC have provided strong support for the hypothesis that accessory folding factors are critical for the biogenesis of SpeB and that the Sec pathway is highly compartmentalized. An important advance has been our discovery of the ExPortal, a unique single microdomain of the cellular membrane specialized to contain the Sec translocons. Thus, virulence proteins are not secreted from S. pyogenes in a circumferential pattern, but rather from a single discrete locus positioned distal to either cell pole. This proposal will continue analysis of SpeB to further explore the role of these factors in secretion and virulence and will probe the structure and organization of the ExPortal to test the hypothesis that the ExPortal functions as a novel organelle to coordinate the trafficking, secretion and folding of virulence proteins in S. pyogenes.